Throughout this application, various publications are referenced by arabic numbers within parentheses. Disclosures of these publications in their entirety are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Varicella-Zoster Virus (xe2x80x9cVZVxe2x80x9d) infects dorsal root ganglia (xe2x80x9cDRGxe2x80x9d), enters latency, and may later reactivate to cause zoster. Studies have detected VZV in specific sites at different stages of infection. VZV DNA is present in the oropharynx (27) and in peripheral blood mononuclear cells (xe2x80x9cPBMCsxe2x80x9d) of patients with chickenpox (3, 16, 20). Virus DNA, the glycoproteins gE and gB, and the immediate-early protein 63 (xe2x80x9cIE63pxe2x80x9d) are found in skin biopsy samples obtained from patients with chickenpox or zoster (1, 23-25). VZV is found in keratinocytes, antigen-presenting cells, and endothelial cells during acute zoster (23, 25) and in keratinocytes and inflammatory cells during chickenpox (1). VZV is present in neurons and satellite cells of DRG years following primary infection (6-8, 12, 17, 22) and has been observed by electron microscopy in sensory nerves during zoster (10). Other details of VZV pathogenesis remain speculative, including how the virus spreads from respiratory epithelial cells to PBMCs, keratinocytes, and DRG. Because PBMCs, sensory nerves, and epithelial cells are in close proximity in the dermis and epidermis, the skin is likely the site where this virus enters the nervous system.
By analogy with herpes simplex virus (xe2x80x9cHSVxe2x80x9d), it is thought that VZV transcription is temporally regulated. Immediate-early (xe2x80x9cIExe2x80x9d) genes are expressed first, followed by early (xe2x80x9cExe2x80x9d) genes and lastly late (xe2x80x9cLxe2x80x9d) genes (5). Some VZV proteins encoded by IE and L genes are incorporated in the virion, including trans-activators such as IE63p and structural proteins such as gC (14, 15). ORF29p (for open reading frame 29 protein), the major VZV DNA-binding protein, is encoded by a putative E gene and is not detected in purified virions (13). ORF29p is also referred to herein as xe2x80x9c29p proteinxe2x80x9d, xe2x80x9cORF29p proteinxe2x80x9d, and xe2x80x9cVZV ORF29p protein.xe2x80x9d During latency, VZV exhibits limited gene expression (6-9, 22), with the accumulation of specific IE and E gene-encoded proteins in neurons (18, 19). During reactivation, all kinetic classes of VZV genes are expressed in neurons (18). Whether VZV is in the lytic or latent state is reflected by the localization of expressed VZV gene products. VZV IE and E proteins that are present in both the nucleus and cytoplasm during productive infection are detected only in the cytoplasm of neurons during latency (18).
Early observations suggested that there were inclusion bodies in endothelial cells present in varicella lesions (29). However, there was no known association between VZV histology and viral etiology at that time.
The present invention provides a first composition of matter comprising 29p protein having bound thereto an agent whose delivery into a eukaryotic cell is desired, which composition of matter enters the cell upon contact therewith.
The present invention also provides a second composition of matter comprising a 29p protein having operably affixed thereto a lipid-soluble moiety which permits the protein to be anchored to a lipid membrane.
The present invention also provides a lipid vesicle comprising the second composition of matter anchored thereto via its lipid-soluble moiety, such that the 29p protein is situated on the vesicle""s outer surface and facilitates delivery of the vesicle""s contents into a eukaryotic cell when the vesicle is contacted therewith. The present invention further provides a monoclonal antibody which specifically binds to 29p protein.
The present invention further provides a method for delivering an agent into a eukaryotic cell comprising contacting the agent with the cell, wherein the agent has bound thereto 29p protein which enters the cell upon contact therewith, thereby delivering the agent into the cell.
The present invention further provides a method for causing a eukaryotic cell to secrete a desired protein in the form of a fusion protein, comprising introducing into the cell a vector for expressing a fusion protein that comprises the desired protein and 29p protein operably affixed thereto, whereby the cell expresses the fusion protein and the 29p protein thereof permits the fusion protein""s exit from the cell, thereby causing the cell to secrete the desired protein in the form of a fusion protein.
The present invention further provides a pharmaceutical composition comprising (a) a composition of matter comprising 29p protein having bound thereto a therapeutic or prophylactic agent, which composition of matter enters a eukaryotic cell upon contact therewith, and (b) a pharmaceutically acceptable carrier.
The present invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a lipid vesicle comprising (a) a therapeutic or prophylactic agent therein, and (b) a 29p protein having operably affixed thereto a lipid-soluble moiety, which protein (i) is anchored to the vesicle via its lipid-soluble moiety, (ii) is situated on the vesicle""s outer surface, and (iii) facilitates delivery of the agent into a eukaryotic cell when the vesicle is contacted therewith.
The present invention further provides a method for treating a subject afflicted with a disorder comprising administering to the subject a therapeutically effective amount of the first or second pharmaceutical composition, wherein the therapeutic agent therein is known to ameliorate the disorder.
The present invention further provides a method for inhibiting the onset of a disorder in a subject comprising administering to the subject a prophylactically effective amount of the first or second pharmaceutical composition, wherein the prophylactic agent therein is known to inhibit the disorder""s onset.
The present invention further provides a nucleic acid molecule which hybridizes to at least a portion of a nucleic acid molecule encoding 29p protein.
The present invention further provides a method for detecting the presence of a 29p protein-encoding nucleic acid molecule in a sample comprising the steps of (a) contacting the sample with the instant detectable nucleic acid molecule under conditions permitting it to hybridize to a 29p protein-encoding nucleic acid molecule if present in the sample, and (b) detecting the presence of any detectable nucleic acid molecule so hybridized, thereby detecting the presence of a 29p protein-encoding nucleic acid molecule in the sample.
Finally, the present invention provides a method for quantitatively determining the amount of 29p protein-encoding nucleic acid molecule in a sample comprising the steps of (a) contacting the sample with the instant detectable nucleic acid molecule under conditions permitting it to hybridize to any 29p protein-encoding nucleic acid molecule present in the sample, (b) quantitatively determining the amount of detectable nucleic acid molecule so hybridized, and (c) comparing this amount to a known standard, thereby quantitatively determining the amount of 29p protein in the sample.